The electrochemiluminescence (ECL) behavior of pyronin derivatives was reported. We found that these derivatives, as novel ECL emitters, generated an efficient emission phenomenon; that is, they showed anodic emission using tri-propylamine (TPA) as the coreactant and cathodic ECL emission using K2S2O8 as the coreactant. Because of its remarkable luminescent performance, the ECL mechanism (reductive-oxidative and oxidative-reductive) of benzylthiol-substituted pyronin (BTP) was specifically studied. Surprisingly, the cathodic ECL emission of BTP had a lower ECL peak potential at −0.54 V (vs. SCE) in aqueous solution. The potential range of −0.7 V to 0.0 V is lower than that of previously reported K2S2O8 cathodic ECL systems. Its good water solubility, easy modification, and excellent emission properties facilitate appealing (bio)analytical applications in ECL biosensors and cell imaging.